Optimization of headspace solid phase microextraction (HS-SPME) for gas chromatography mass spectrometry (GC–MS) analysis of aroma compounds in cooked beef using response surface methodology

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Optimization of headspace solid phase microextraction (HS-SPME) for gas chromatography mass spectrometry (GC–MS) analysis of aroma compounds in cooked beef using response surface methodology
  1088 Plant Disease / Vol. 98 No. 8 Optimization of Headspace Solid-Phase Microextraction Conditions for the Identification of Phytophthora cinnamomi   Rands Rui Qiu,  College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi 712100, China; Cooperative Research Centre for National Plant Biosecurity, Bruce, ACT 2617 Australia; and School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA, 6150 Australia; Dong Qu,  College of Natural Resources and Environment, Northwest A&F University, Yangling; Giles E. St. J. Hardy,  Centre for Phytophthora Science and Management (CPSM), School of Veterinary and Life Sci-ences, Murdoch University, Murdoch, WA, 6150 Australia; and Cooperative Research Centre for National Plant Biosecurity, Bruce;   Robert Trengove and Manjree Agarwal,  Cooperative Research Centre for National Plant Biosecurity, Bruce; and School of Veterinary and Life Sciences, Murdoch University, Murdoch; and Yonglin Ren,  Cooperative Research Centre for National Plant Biosecurity, Bruce; School of Veterinary and Life Sciences, Murdoch University, Murdoch; and Department of Agriculture and Food, Western Australia, Perth, WA 6151 Australia Abstract Qiu, R., Qu, D., Hardy, G. E. St. J., Trengove, R., Agarwal, M., and Ren, Y. 2014. Optimization of headspace solid-phase microextraction condi-tions for the identification of Phytophthora cinnamomi  Rands. Plant Dis. 98:1088-1098. A robust technique was developed to identify Phytophthora cinnamomi  using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) coupled to a flame ionization detector (FID) for analyzing volatile organic compounds (VOCs). Six fiber types were evaluated and results indicated that the three-phase fiber 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/ PDMS) had the highest extraction efficiency for both polar and nonpo-lar GC columns. The maximum extraction efficiency (equilibrium absorption) was achieved 16 h after fiber exposure in the HS. Absorbed compounds on the fiber were completely desorbed in the GC injector after 5 min at 250°C. Compared with the nonpolar column, the polar column showed optimum separation of VOCs released from P. cinnam-omi . Under the optimized HS-SPME and GC/FID conditions, lower detection limits for the four external standards was found to be be-tween 1.57 to 27.36 ng/liter. Relative standard deviations <9.010% showed that the method is precise and reliable. The method also showed good linearity for the concentration range that was analyzed using four standards, with regression coefficients between 0.989 and 0.995, and the sensitivity of the method was 10 4  times greater than that of the conventional HS method. In this study, the VOC profiles of six Phytophthora  spp. and one Pythium  sp. were characterized by the optimized HS-SPME-GC method. The combination of the VOCs cre-ates a unique pattern for each pathogen; the chromatograms of differ-ent isolates of P. cinnamomi  were the same and the specific VOC pat-tern of P. cinnamomi  remained consistently independent of the growth medium used. The chromatograms and morphological studies showed that P. cinnamomi released specific VOCs at different stages of colony development. Using the optimized HS-SPME GC method, identifica-tion of P. cinnamomi  from 15 in vivo diseased soil samples was as high as 100%. Results from this study demonstrate the feasibility of this method for identifying P. cinnamomi  and the potential use of this method for physiological studies on P. cinnamomi . Phytophthora cinnamomi is a microscopic soilborne plant patho-gen that can invade and destroy the root systems of susceptible native and introduced plant species. It is the most widely distrib-uted  Phytophthora sp., with over 3,000 host species (15). The most significant crop losses due to P. cinnamomi root rot occur in avo-cado and chestnut. P. cinnamomi has been associated with the widespread mortality of oak trees and is the cause of one of the most extensive epidemics in natural ecosystems in the southwest area of Western Australia (4,26). A range of fauna are affected by P. cinnamomi -induced changes to vegetation, including small mammals, reptiles, birds, and invertebrates (12,32). P. cinnamomi , with its enormous host range spanning many genera of rare and endemic plants, has been named by the Australian Government as one of the 15 “Key Threatening Processes” endangering ecosys-tems and ecological processes in Australia (9). Developing robust, timely, and sensitive diagnostic methods for P. cinnamomi  infection is very important to prevent its introduction and spread. Traditionally, identification of Phytophthora  spp. start with isolation, and the main method to isolate Phytophthora spp.   from symptomatic plants is by plating necrotic material onto Phy-tophthora- selective agar or baiting diseased tissues and rhizosphere soil with fresh young leaf material as “baits”; necrotic lesions from the baits are then plated onto selective agar (10). This process, though effective, is slow and requires experienced personnel to identify the pathogen based on morphological characteristics. Elec-trophoretic patterns of isozymes and serological methods based on use of antibodies are only useful for P. cinnamomi  identification from pure cultures and these methods are slow and expensive, have limited usefulness, are not necessarily species-specific, and need to be confirmed by morphological or molecular methods (6). More recently, a variety of molecular techniques have been used for the identification   of P. cinnamomi  and other Phytophthora  spp. Con-ventional polymerase chain reaction (PCR) identification is based on amplification and pyrosequencing (30); fingerprinting tech-niques that include restriction fragment length polymorphism of the internal transcribed spacer region (5,21), random amplified polymorphic DNAs (20), and amplified fragment length polymor-phisms (8) with nested PCR are all based on conventional PCR and are sensitive and simple but require an agarose or acrylamide gel electrophoresis to visualize the amplified bands, and increase the time required for sample analysis. Real-time PCR eliminates the requirement for post-amplification amplicon detection procedures, including gel electrophoresis, and avoids the problem of cross contamination of reactions that is inherent in nested PCR (11). The technique can also be implemented in the field by using portable real-time PCR machines. Also, without the addition of ethidium Corresponding author: Y. Ren, E-mail: y.ren@murdoch.edu.au R. Qiu, G. Hardy, M. Agarwal, and Y. Ren contributed equally to this work.Accepted for publication 4 February 2014. http://dx.doi.org/10.1094/PDIS-12-13-1258-RE  © 2014 The American Phytopathological Society  Plant Disease / August 2014 1089 bromide, health risks for operators and environmental contamina-tion are reduced (29,31). However, it is restricted by the number of fluorescent probes and, in some cases, the primers in multiplex reactions interfere with each other, resulting in a lower sensitivity of detection or the generation of additional amplicons (18,28). Therefore, there is an important need to develop other robust, rapid, highly specific, and cost effective diagnostic methods for the identification of Phytophthora spp. from plant and soil material. A novel nondestructive and noninvasive extraction method (no preprocessing of the sample before separation is required, ensuring sample integrity) for the detection of specific microbial-emitted volatile organic compounds (VOCs) has been developed to dis-criminate microbes in soil, food, feeds, and grains (17,23,25), and a number of studies have shown that VOCs released by microbes change with the different stages of their growth (13,22,34). Although several marker volatiles of P. infestans  have been identi-fied (7,19) and applied to detect P. infestans  infection, specific volatiles have not been discriminated for the identification of P. cinnamomi . Solid-phase microextraction (SPME) is a new sample enrichment and solvent-free extraction technique which integrates sampling, extraction, concentration, and sample introduction in a single step. Since its development in 1989 by Arthur and Pawliszyn (2), its applications have dramatically increased. The SPME in combination with headspace (HS) analysis by gas chromatography (GC; a very common laboratory analytical instrument) is a conven-ient alternative method for the analysis of VOCs. The objectives of this article were to prove the feasibility of us-ing VOCs together with HS-SPME as an identification tool and to establish optimal HS-SPME and GC conditions for the identifica-tion of   P. cinnamomi . Materials and Methods Reagents. BBL agar, grade A (reference 212304), used for preparation of V8 juice agar (V8A), and Difco potato dextrose agar (PDA; reference 213400) were purchased from Becton, Dickinson and Company. Organic solvents, including methanol, ethanol, and ethyl formate, were purchased from MERK (high-performance liquid chromatography [HPLC] grade); hexane was purchased from Fisher Scientific (catalog number H306, HPLC grade). Equipment and apparatus. Erlenmeyer flasks (100 ml; Quickfit, catalog number QFY-372-P) were used for preparation of samples. Each flask was fitted with an adapter (Quickfit, part num-ber AQST53/13) equipped with a septum (Grace, catalog number 6518). Glass bottles of 250 ml (Alltech, catalog number 9535), each fitted with a Mininert valve and septum (Alltech, catalog number 95326) were used for preparation of gas standards. An Olympus BX51 microscope (serial number 3M08876) was used to study the morphology of P. cinnamomi . Six different types of SPME fiber were compared to evaluate their effectiveness on the extraction of VOCs produced by P. cin-namomi . The tested SPME fibers were 7 µm polydimethylsiloxane (PDMS) fiber (Sigma-Aldrich Australia, catalog number 57307), 100 µm PDMS fiber (Sigma-Aldrich Australia, catalog number 57300-U), 50/30 µm divinylbenzene/carboxen (DVB/CAR)/PDMS fiber (Sigma-Aldrich Australia, catalog number 57348-U), 65 µm PDMS/DVB fiber (Sigma-Aldrich Australia, catalog number 57310-U), 85 µm polyacrylate (PA) fiber (Sigma-Aldrich Australia, catalog number 57307), and 85 µm CAR/PDMS fiber (Sigma-Aldrich Australia, catalog number 57334-U). Prior to use, the fi-bers were conditioned according to the manufacturers’ recommen-dations, and cleaned between each extraction by exposing the fibers into the GC injection port for 15 min at 250°C for the 100 µm PDMS and PDMS/DVB fibers, and 15 min at 260°C for the other four fibers. Analysis of VOCs was performed on an Agilent 6890 series (se-rial number US00021731) gas chromatograph coupled to a flame ionization detector (FID). Injector and detector temperatures were 250°C, except when the injector temperature was varied to assess its effect on desorption of VOCs from the fiber. In order to get the optimal blueprint of P. cinnamomi , both polar and nonpolar col-umns were used for the separation of VOCs. Separation was achieved on a ZB-WAX plus capillary polar column (30 m by 0.25 mm ID, film thickness 0.25 µm; Zebron, part number 7HG-G013-11) and an Rxi-5ms nonpolar column (30 m by 0.25 mm, film thickness 0.25 µm; Crossbond 5% diphenyl and 95% dimethyl polydiloxane; RESTEK, catalog number 13423) separately. Hydro-gen was used as carrier gas at a flow rate of 1.0 ml min –1 . The GC column temperature was programmed to hold temperature at 45°C for 5 min, then increased by 5°C min –1  to 250°C and held for 5 min, and the GC-FID instrument was operated under the splitless mode. Data were collected with GC online software and analyzed by ChemStation instrument offline software and Microsoft Excel 2007.  Phytophthora   and    Pythium   spp.,    P. cinnamomi  isolates, VOC detection, and microscopic observation.  Six Phytophthora  spp., including five isolates of P. cinnamomi (Table 1), P. niederhauserii (PAB 13-29), P.   elongata (VHS 13784), P.   humicola (VHS 25241), P.   inundata (VHS 25170), and P.   multivora (VHS 14926); and one Pythium  sp., Pythium dissotocum (SA370), were obtained from the Centre for Phytophthora Science and Management (CPSM), Mur-doch University, Australia. Phytophthora and Pythium spp. were grown on 10% V8A in pe-tri dishes in the dark at 24 ±  1°C and subcultured every 10 days via mycelial mass transfer. A single 4-mm-diameter plug from the edge of the 5-day-old pathogen cultures was used as inoculum and transferred to a 100-ml Erlenmeyer flask containing 50 ml of V8A. Inocula of the five Phytophthora cinnamomi  isolates were also transferred to 50 ml of PDA. All the cultures were incubated at 24 ±  1°C in the dark for 6 days and VOC detection was conducted. A single 4-mm-diameter agar disc colonized by a 5-day-old P. cinnamomi isolate (MP94.48) colony grown on V8A   was trans-ferred to a 100-ml Erlenmeyer flask containing 50 ml of V8A, the culture was incubated for 15 days at 24 ±  1°C in the dark, and morphological observation and VOC detection were conducted daily. Each treatment combination was replicated three times and each experiment was repeated more than three times. Inoculation. Seedlings of  Lupinus angustifolius  ‘Danja’, the commonly used bait plant for P. cinnamomi , were used as the sus-ceptible host plant   for this study. Seed of  L. angustifolius  Danja were surface sterilized by soaking them with 70% ethanol for 2 min followed by immersion in 50% bleach solution (6.25% availa-ble chlorine) for 5 min, and then rinsed five times in sterile dis-tilled water. The sterilized seed were germinated on sterilized filter paper, which was premoistened with sterilized distilled water at 24 ±  1°C in the dark for 2 days.  Lupin sample inoculation.  Three uniform 2-day-old lupin seed-lings were transferred to a 100-ml Erlenmeyer flask with 1 ml of sterilized distilled water and grown for a further day. A single 4-mm plug from the edge of the 5-day-old P. cinnamomi  MP 94.48 Table 1.   Phytophthora cinnamomi  isolates used in this study a   Isolate code Type GenBank number Country Host Year MP 94.48 A2 JX113294 Australia  Eucalyptus marginata  1994 W4 A2 JX113312 Australia Chamaescilla corymbosa  2011 W15 A2 JX113308 Australia Stylidium diuroides  2011 MP75 A1 NA Australia  Eucalyptus marginata  ND DCE 25 A1 JX454790 Australia  Hypocalymma angustifolia  1969 a NA = not applicable, and ND = no data.  1090 Plant Disease / Vol. 98 No. 8 culture was used as inoculum, placed on the tip of the root, and kept in dark at 24 ±  1°C for 3 days to allow lesions to develop. Soil sample inoculation.  Washed white river sand was purchased from Sand and Soil. To ensure that the seedlings were grown at “container capacity” conditions for optimum growth, 13.6 ml of 10% V8 broth was added to a 100-ml Erlenmeyer flask containing 100 g of sand and sterilized. Three lupin seedlings were transferred to the sterilized sand and grown for a further 2 days. Six 4-mm-diameter agar plugs colonized with 5-day-old P. cinnamomi MP94.48 were transferred aseptically to the middle of the sand layer in each flask; controls consisted of six similar-sized V8A plugs that had not been colonized by the pathogen. All the treat-ments were then incubated for 6 days at 24 ±  1°C in the dark, at which time VOC detection was conducted. Optimization of HS-SPME. According to Risticevic et al. (24), a typical SPME method can be optimized for many aspects and, based on the objectives of the current study, the following SPME parameters were evaluated and optimized: fiber coating, extraction time, and desorption conditions. While optimizing one condition, all other conditions were kept constant. Some parameters such as the VOC collection chamber (100-ml Erlenmeyer flask with an adapter and a septum), 250°C GC detector temperature, and the fiber extraction temperature of 24 ±  1°C were kept constant throughout the experiment. The sensitivity of an HS-SPME in combination with GC relies on optimized conditions. The first step followed in this study was to optimize the HS-SPME method to maximize the range and amount of VOCs extracted. An optimized chromatographic separa-tion begins with the column: a nonpolar column is better for anal-yses of nonpolar compounds while polar columns most effectively separate polar compounds. In order to get the most reliable infor-mation, both polar and nonpolar columns and polar and nonpolar fibers were used in this study. Selection of fiber.  The six fiber types were exposed in the HS of a 100-ml Erlenmeyer flask over the 5-day-old P. cinnamomi  MP 94.48 colony grown on V8A and V8A alone (as control) for 3 h of exposure and 5 min of desorption in the GC injector.  Determination of exposure time of fiber extraction.  The selected fiber was exposed to the HS of the 100-ml flasks containing a 5-day-old P. cinnamomi  MP 94.48   colony grown on 50 ml of V8A and V8A alone for a time periods of 3 to 24 h for both the polar column and nonpolar column. After exposure, the fiber was retrieved and injected into the heated GC injection port (250°C) and desorbed for 5 min. For each treatment and time combination, there were three replicate flasks and three replicate extractions per flask.  Determination of desorption temperature and time.  The selected fiber (50/30 µm DVB/CAR/PDMS) and the optimized fiber extrac-tion time (16 h) were used to optimize the GC injector temperature and desorption time. Determination of the optimal injector tem-perature was conducted on both the polar and nonpolar columns. The fiber was desorbed in the GC injection port at different injec-tor temperatures (from 200 to 270°C; 270°C is the highest tem-perature the fiber can withstand) for 5 min. The fiber was desorbed for different desorption times (1, 3, 5, and 7 min) at the selected optimal injector temperature to determine the optimal desorption time. Fig. 1.  Peaks produced by Phytophthora cinnamomi growing on V8 juice agar and V8 juice agar alone extracted by six different fiber types that were separated by a polar column (ZB-WAX plus) and a nonpolar column (Rxi-5ms). Table 2. Soil samples used to detect the presence of Phytophthora cinnamomi  in this study Sample code Host a  Location 716  Banksia attenuata 30°38 ′ 02 · 6 ′′ S, 115°25 ′ 45 · 3 ′′ E 717  Banksia menziesii 30°38 ′ 02 · 3 ′′ S, 115°25 ′ 45 · 5 ′′ E 722 Trachymene sp.; Ericaceae (unid.)   30°37 ′ 52 · 8 ′′ S, 115°25 ′ 58 · 3 ′′ E T2+4  Hibbertia sp.   30°38 ′ 02 · 7 ′′ S, 115°25 ′ 48 · 2 ′′ E T2+6  Banksia attenuata ;  Myrtaceae  (unid.)   … T3+4  Hibbertia sp.;  Melaleuca sp.   30°37 ′ 59 · 2 ′′ S, 115°25 ′ 47 · 8 ′′ E T3+6  Banksia attenuata , Conostephium sp., Ericaceae (unid.)   … T3+8 Ericaceae (unid.);  Banksia  sp. (unid.) roots … T6-4 Ericaceae (unid.) 30°38 ′ 02 · 7 ′′ S, 115°25 ′ 45 · 6 ′′ E MU22-1 Calytrix sp.;  Banksia  sp. (unid) roots 32°04 ′ 39 · 4 ′′ S, 115°49 ′ 56 · 4 ′′ E MU22-2  Hibbertia sp.;  Banksia  (unid.) roots … MU22-3  Brachyloma    preissii  … MU22-4  Banksia  sp. (unid.) roots and soil … MU22-5  Brachyloma    preissii  … MU04 Stirlingia latifolia  32°04 ′ 28 · 5 ′′ S, 115°50 ′ 32 · 4 ′′ E a Unid. = unidentified family or genus.  Plant Disease / August 2014 1091 Selection of GC column.  The separation efficiency of the polar and nonpolar columns was evaluated with the optimized three-phase fiber at the optimal time of extraction and time and tempera-ture of desorption. Determination of the limit of detection for the HS-SPME coupled to GC-FID method. The limit of detection   (LOD) was evaluated with hexane, methanol, ethanol, and ethyl formate as external standards. For preparation of the standards, the appropriate volumes of each standard were added into a sealed 250-ml bottle. After 5 min of extraction with the fiber 50/30 µm DVB/CAR/ PDMS at room temperature (24 ±  1°C), the fiber was injected into the gas chromatograph at the injector temperature of 250°C. All samples were duplicated and each sample were analyzed as triplicate injections. Identification of   P. cinnamomi  from forest soil samples using the optimized HS-SPME GC method.   Fifteen soil samples natu-rally infected by P. cinnamomi  were collected from diseased, sus-ceptible plant rhizosphere from Murdoch University campus and sites south of Perth (Table 2). The youngest fully expanded oak ( Quercus suber  ) leaves (Kings Park, WA) were used as the baits for P. cinnamomi . All the soil samples were separated into two groups; one group was used for HS-SPME GC analysis and the other group used for baiting. Soil samples for baiting were transferred into 1-liter takeaway containers and filled with deionized water. The fresh oak leaves were then floated on the water surface. These baits were checked every 24 h for 10 days, and any leaves that developed water-soaked lesions were removed with forceps and placed into a 100-ml Erlen-meyer flask in preparation for HS-SPME extraction. In addition to the above, some of the soaked baits were dried on paper towels; then, small sections approximately 5 mm square were excised with a sterile scalpel and plated onto Phytophthora  selective medium (NARPH; 16) and the plates were incubated at 24 ±  1°C in the dark. The plates were checked daily under the compound micro-scope to determine whether P. cinnamomi  had been successfully isolated from the soil. Baiting water (50 ml) for each soil sample was filled into a 100-ml Erlenmeyer flask in preparation for HS-SPME extraction also. In addition to baiting, 50 g of each soil sample was placed di-rectly into a 100-ml Erlenmeyer flask for HS-SPME extraction. The optimized HS-SPME GC method was used for analyzing the VOCs from fresh soil, baiting water, and baits. Statistical analysis. The variations (standard errors of the mean) of VOCs and external standards concentrations, the triplicate sam-ples, and the triplicate injections in comparison with average read-ings were analyzed by Microsoft Excel 2007. Results Selection of fiber and determination of extraction time. The extraction efficiency of the six different commercially available SPME fibers (7 and 100 µm PDMS, DVB/CAR/PDMS, PDMS/DVB, PA, and CAR/PDMS) was evaluated by comparing Fig. 2. A,  Effects of extraction time with different fibers on the extraction efficacy of the total compounds identified from Phytophthora cinnamomi   + V8 juice agar at 3, 6, 9, 12, 16, and 20 h for the nonpolar column (Rxi-5ms); B, effects of extraction time with the 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber on theextraction efficiency of seven analytes of interest (>85% of total identified volatile organic compounds) at 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h for the polar column (ZB-WAX plus). Bars represent standard errors of mean ( n  = 3).  1092 Plant Disease / Vol. 98 No. 8 the peak numbers of the compounds from P. cinnamomi  + V8A and V8A alone treatments under the same extraction, desorption, and GC conditions. After separation of VOCs desorbed from different fibers on both polar and nonpolar columns, the results showed that the three-phase fiber (50/30 µm DVB/CAR/PDMS fiber) had the highest extraction efficiency (Fig. 1). The GC response of fiber 50/30 µm DVB/CAR/PDMS after 16 h of extraction was more than two times that of fiber 85 µm PA and four times that of fiber 85 µm CAR/PDMS when separated on the nonpolar column (Fig. 2A), and trapped a wider range of VOCs that separated on both the polar and nonpolar columns (Fig. 1). There were no VOCs extracted with the 7 µm PDMS fibers because no compounds separated from both polar and nonpolar columns (Figs. 1 and 2A). Therefore, the three-phase fiber was selected as the appropriate fiber to optimize the other parameters. For determination of extrac-tion time, the selected three-phase fiber was exposed in the HS of the flask for different times (3 to 24 h). The levels of desorbed compounds increased with increasing extraction time until 16 h on the nonpolar column (Fig. 2A) and polar column (Fig. 2B), when equilibrium was achieved. Evaluation of GC injector (fiber desorption) temperature and desorption time.   The effect of the GC injector temperature was evaluated with the selected 50/30 µm DVB/CAR/PDMS fiber at GC inlet temperatures of 200 to 270°C under the HS fiber ex-traction time of 16 h over P. cinnamomi  + V8A and V8A alone. The results indicated that the total area of desorbed compounds detected increased with increasing injector temperature for both the polar and nonpolar columns, and the maximum desorption from the fiber was obtained at 250°C (Fig. 3A). The total desorbed com-pounds produced decreased when the temperature was greater than 250°C. The various desorption times had a significant effect on GC re-sponse of P. cinnamomi  + V8A and V8A alone. The total area of desorbed VOCs increased with an extension of desorption time from 1 to 5 min and then reached equilibrium at 5 min; the GC response remained constant after a desorption time of 5 min, which was determined as the optimum (Fig. 3B). Selection of column. The three-phase fiber was exposed in the HS over a 6-day-old P. cinnamomi  + V8A colony for 16 h and separated on both polar and nonpolar columns under the optimized HS-SPME GC conditions. The VOCs of the samples had better separation and were more sensitive for most separated VOCs on the polar column than the nonpolar column, and a broader range of compounds was captured on the polar column (Fig. 4). Therefore, the polar column was selected for the subsequent studies. Fig. 3. A,  Effects of gas chromatography (GC) injector temperature on desorption of volatile organic compounds (VOCs) of Phytophthora cinnamomi   + V8 juice agar and V8 uice agar alone from the 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber after 16 h of extraction; B,  effects of desorption time on desorption of VOCs of P. cinnamomi   + V8 juice agar and V8 juice agar alone from the 50/30 µm DVB/CAR/PDMS fiber. Bars represent standard errors of mean ( n  = 3).
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